Polyethylene glycol significantly enhances the transfer of membrane immunoblotting.
Poly(ethylene glycol)n is a group of water-soluble, hydrophobic, optically transparent and biomacromolecule-nondenaturing polymers. These properties have caused it be widely used for various purposes in the biological sciences. In this study, the effects of poly(ethylene glycol)n on protein preservation, electrotransferring, and immunoblotting from sodium dodecyl sulfate (SDS)-polyacrylamide gel onto polyvinylidene difluoride (PVDF) membrane have been systematically evaluated. After SDS-polyacrylamide gel electrophoresis, 30% poly(ethylene glycol)n may be applied to reversibly fix proteins within the gel more completely, differing from irreversible fixation produced by solutions such as trichloroacetic acid-sulfosalicylic acid or acetic acid-methanol systems. The intragel proteins, fixed by poly(ethylene glycol)n, can be electroblotted directly onto PVDF membranes in the presence of 30% poly(ethylene glycol)n. We have shown that treatment with poly(ethylene glycol)n may reduce background, raise signal-to-noise ratio, sharpen protein bands, and increase resolution, resulting in enhancement of the immunoblotting transfer. It is possible to visualize a few picograms of a single protein band, increasing the sensitivity of the method by 10- to 100-fold, as compared with standard immunoblotting techniques.
Zeng
C
C
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.
Suzuki
Y
Y
Alpert
E
E
D000818:Animals; D001798:Blood Proteins / Q000032:analysis; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008565:Membrane Proteins / Q000032:analysis; D008567:Membranes, Artificial; D011092:Polyethylene Glycols; D011145:Polyvinyls; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate; D005465:Fluorocarbon Polymers
D001798:Blood Proteins; D008565:Membrane Proteins; D008567:Membranes, Artificial; D011092:Polyethylene Glycols; D011145:Polyvinyls; D012967:Sodium Dodecyl Sulfate; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers